Name: ultrahigh resolution mouse optical coherence tomography to aid intraocular injection in retinal gene therapy research
Uploaded: 2018-11-02T12:30:00
Description: Watch this Scientific Journal Video about Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research at JoVE.com

This material is based upon work supported, in part, by the Department of Veterans Affairs (VA), Veterans Health Administration, Office of Research and Development (Biomedical Laboratory Research and Development) (VA Merit Grant 1I01BX000669). JMS is employed, in part, as Staff Physician-Scientist, Ophthalmology, by VA WNY; MCB is employed, in part, by VA WNY. The study was conducted at, and supported in part by, the Veterans Administration Western New York Healthcare System (Buffalo, NY). Contents do not represent the views of the Department of Veterans Affairs or the United States Government. Also supported, in major part, by NIH/NEI R01 grant EY013433 (PI: JMS), NIH/NEI R24 grant EY016662 (UB Vision Infrastructure Center, PI: M Slaughter, Director- Biophotonics Module: JMS), an Unrestricted Grant to the Department of Ophthalmology/University at Buffalo from Research to Prevent Blindness (New York, NY), and a grant from the Oishei Foundation (Buffalo, NY). We acknowledge the gift of the hC1 transgenic RHOP347S&#160;line and the exon 1 mouse RHO knockout from Dr. Janis Lem (Tufts New England Medical Center, Boston, MA), and the gift of the NHR-E transgene model in the heterozygous state on the mouse exon 2 RHO knockout background from Drs. G. Jane Farrar and Peter Humphries (Trinity College, Dublin, IRE).

Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committees of the VA WNY HCS and the University at Buffalo-SUNY. Animals were used according to the stipulations of the Association for Research in Vision and Ophthalmology (ARVO) and the Declaration of Helsinki.
1. Mouse Models
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	<li>Identify mouse models to be evaluated including controls. 
	NOTE: Imaging was performed for a C57BL/6(J), hC1/hC1//mWT/mWT, a partially humanized mous...

<ol>
	<li>Regus-Leidig, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+knockdown+of+Piccolino+disrupts+presynaptic+ribbon+morphology+in+mouse+photoreceptor+synapses.">In-vivo knockdown of Piccolino disrupts presynaptic ribbon morphology in mouse photoreceptor synapses.</a> Front Cell Neurosci. 8 (259), 1-13 (2014).</li><li>Jiang, L., Frederick, J. M., Baehr, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+gene+therapy+in+dominant+retinitis+pigmentosa+and+cone-rod+dystrophy+mouse+models+caused+by+GCAP1+mutations.">RNA interference gene therapy in dominant retinitis pigmentosa and cone-rod dystrophy mouse models caused by GCAP1 mutations.</a> Front Mol Neurosci. 7 (25), 1-8 (2014).</li><li>Seo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+gene+therapy+of+mice+with+Bardet-Beidl+Syndrome+Type-1.">Subretinal gene therapy of mice with Bardet-Beidl Syndrome Type-1.</a> Invest. Ophthalmol. Vis. Sci. 54 (9), 6118-6132 (2013).</li><li>Molday, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RD3+gene+delivery+restores+guanylate+cyclase+localization+and+rescues+photoreceptors+in+the+RD3+mouse+model+of+Leber+congenital+amaurosis+12.">RD3 gene delivery restores guanylate cyclase localization and rescues photoreceptors in the RD3 mouse model of Leber congenital amaurosis 12.</a> Hum. Mol. Genet. 22 (19), 3894-3905 (2014).</li><li>Pang, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV-mediated+gene+therapy+in+mouse+models+of+recessive+retinal+degeneration.">AAV-mediated gene therapy in mouse models of recessive retinal degeneration.</a> Curr. Mol. Med. (3), 316-330 (2012).</li><li>Vandenberghe, L. H., Auricchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+adeno-associated+viral+vectors+for+retinal+gene+therapies.">Novel adeno-associated viral vectors for retinal gene therapies.</a> Gene Ther. 19 (2), 162-168 (2012).</li><li>Tenenbaum, L., Lehtonen, E., Monahan, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+risks+related+to+the+use+of+adeno-associated+virus-based+vectors.">Evaluation of risks related to the use of adeno-associated virus-based vectors.</a> Curr. Gene Ther. 3 (6), 545-565 (2003).</li><li>Parikh, S., Le, A., Davenport, J., Gorin, M. B., Nusinowitz, S., Matynia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+and+validated+injection+method+for+accessing+the+subretinal+space+via+a+transcleral+posterior+approach.">An alternative and validated injection method for accessing the subretinal space via a transcleral posterior approach.</a> J. Vis. Exp. (118), e54808 (2016).</li><li>Bainbridge, J. W. B., Mistry, A. R., Thrasher, A. J., Ali, R. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+injection+of+gene+therapy+vectors+and+stem+cells+in+the+perinatal+mouse+eye.">Subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye.</a> J. Vis. Exp. (69), e4286 (2012).</li><li>Berger, A., al, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral-domain+optical+coherence+tomography+of+the+rodent+eye:+highlighting+layers+of+the+outer+retina+using+signal+averaging+and+comparison+with+histology.">Spectral-domain optical coherence tomography of the rodent eye: highlighting layers of the outer retina using signal averaging and comparison with histology.</a> PLoSOne. 9 (5), 96494 (2014).</li><li>Muhlfriedel, R., Michalakis, S., Garrido, M. G., Biel, M., Seeliger, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+technique+for+subretinal+injections+in+mice.">Optimized technique for subretinal injections in mice.</a> Methods in Molecular Biology. , (2013).</li><li>Sarra, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+transgene+expression+in+mouse+retina+following+subretinal+injection+of+recombinant+adeno-associated+virus.">Kinetics of transgene expression in mouse retina following subretinal injection of recombinant adeno-associated virus.</a> Vision Res. 42, 541-549 (2002).</li><li>Yan, Q. 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HR-SD-OCT provides a simple method for characterization of potential animal models of human disease to determine their usefulness in testing potential therapeutics. The ability to quickly and reliably characterize a potential animal model of human disease is critical to the process of therapeutic drug discovery (e.g., replacement gene therapy, ribozyme or shRNA knockdown gene therapy, combined gene therapy). HR-SD-OCT provides a simple, quick, and non-invasive method for evaluating retinal health that can be use...

Researchers are developing gene therapies for a variety of retinal and retinal degenerative diseases with hopes of translating novel therapeutics into treatments for human disease1,2,3,4,5,6,7,8,9,10,11. Time domain or spectral domain optical coherence tomography (SD-OCT) has been used to investigate the aspects of outer retinal degeneration in specific mouse models of disease12,13,14. HR-SD-OCT has not, however, been extensively used in the context of optimizing evaluation of mouse models to determine the rate and spatial uniformity of retinal degeneration, or in the context of preclinical evaluation of gene based therapeutics, for example, to assess rescue, toxicity, or the spatial extent of vector delivery8,15,16. Once a mouse model is fully characterized, the HR-SD-OCT data can serve as an informative and reliable resource to measure the impact of therapeutics to exert rescue or toxicity in mouse models of retinal degeneration17. Many groups are using subretinal injection as a method of vector delivery due to its efficiency at transducing photoreceptors and retinal pigment epithelium (RPE) cells. However, this remains a difficult method to master, given that it is typically done by free-hand surgery from the corneal surface, and is often fraught with cataracts, bleeding, and unintended retinal detachments occurring simply by manipulation of the posterior vitreous. Many groups still attempt subretinal injections blindly and deliver the virus using manual injections with relatively large diameter stainless steel needles (34G)8,17,18,19,20,21,22, and a few uses optical coherence tomography (OCT) imaging to confirm proper delivery of vector to the retina8,17,20,22. Some improvements in the method have recently been described using microscale needles driven by a micromanipulator22.
We present an integrated approach which aids in the positioning of the needle, and the injections are facilitated by a custom directed stereo ophthalmoscope designed in the lab specifically for visualizing inside the small eye of the mouse17,23. The use of pulled glass micro needles in conjunction with the stereotaxic micromanipulator provide better control of needle placement with no surgical cut down required (i.e., through conjunctivae and connective tissue) prior to injection. The use of the pressure regulated micro injector helps deliver consistent injection volumes, and the injection can be done with much greater stability, precision, and much slower than manual injections performed by a hand-held syringe, thereby decreasing the occurrence of bubble injection into the eye. The smaller needle helps prevent leakage following needle withdrawal because the path is self-sealing. To assess the extent of injection/delivery, many investigative groups rely on finding and assessing the areal extent of enhanced green fluorescence protein (EGFP) expression in the retina (expression construct delivered by the vector) at the experimental end point (euthanasia) to confirm successful injections11,19,20,24. This approach (not utilizing OCT) to verify surgical success wastes an enormous amount of resources in surgical procedural time and animals, since all animals with (unknown) surgical failures need to be maintained, followed with repetitive measures until euthanasia and eye harvest (when EGFP is measured). Confirmation of the location of injection in the retina can be improved using HR-SD-OCT to demonstrate that the injection is located between the correct layers of the retina (i.e. the subretinal space). HR-SD-OCT can also be used to immediately delineate unsuccessful attempts (surgical failures) to identify relevant variables in real surgical time to improve upon the approach. We found that HR-SD-OCT provides numerous advantages in preclinical gene therapy studies by allowing rapid quantitative evaluation of outer retinal degeneration, allowing identification/culling of study animals which do not meet experimental criteria (e.g., incorrect subretinal injection), and to direct follow-up imaging to the region of the eye where vector was delivered (where preclinical effect is most likely) as well as control regions where vector was not delivered. Since its development, the use of SD-OCT has continued to be accepted and used by ophthalmology researchers and is now considered the standard of retinal imaging in retinal scientific studies in mouse or rodent models13,25. HR-SD-OCT and its software capabilities were utilized in unique integrated ways to further the goal of successful quantitative gene therapy in mouse models at every step in the process, including animal model selection, characterization of degeneration in chosen disease models, therapeutic delivery, mapping of vector delivery, and toxicity/efficacy evaluation. The use of HR-SD-OCT allows for more efficient drug discovery at every level of the process. Here we describe these approaches that are used in our RNA Drug Discovery program.

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			<td height="39" style="height:39px;width:251px;">C57BL6 (J)</td>
			<td style="width:245px;">Jackson Laboratories</td>
			<td style="width:215px;">664</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">N129R-</td>
			<td style="width:245px;">N/A</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">2HRho 1T/1T</td>
			<td style="width:245px;">N/A</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="78" style="height:78px;width:251px;">Envisu R-2200 Ocular Coherence Tomography Instrument (OCT)</td>
			<td style="width:245px;">Bioptigen</td>
			<td style="width:215px;">90-R2200-U1-120.&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Retinal Imaging System (RIS)</td>
			<td style="width:245px;">In-house</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Stemi 2000C Microscope</td>
			<td style="width:245px;">Zeiss</td>
			<td style="width:215px;">000000-1106-133</td>
			<td></td>
		</tr>
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			<td height="39" style="height:39px;width:251px;">P-97 Flaming/Brown micropipette puller</td>
			<td style="width:245px;">Sutter Instrument Co</td>
			<td style="width:215px;">p-97</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">MMN-33 micro manipulator&#160;</td>
			<td style="width:245px;">Narishige USA</td>
			<td style="width:215px;">MMN-33</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">PLI-100&#160; micro injector</td>
			<td style="width:245px;">Harvard Apparatus</td>
			<td style="width:215px;">64-1736</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Micropipette Holder (Rotating)</td>
			<td style="width:245px;">In-house</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Micropipette Storage Receptacle</td>
			<td style="width:245px;">World Precision Instruments Inc.</td>
			<td style="width:215px;">E210</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">&#160;Borosilicate glass capillary tubes 1.5-1.8 X 100mm,&#160;</td>
			<td style="width:245px;">Harvard Apparatus</td>
			<td style="width:215px;">30-0053</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">2,2,2-Tribromoethanol</td>
			<td style="width:245px;">SIGMA Aldrich</td>
			<td style="width:215px;">T48402-25G</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Tert-amyl Alcohol</td>
			<td style="width:245px;">SIGMA Aldrich</td>
			<td style="width:215px;">240486-100ML</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Atropine Sulfate Ophthalmic Solution, USP 1%</td>
			<td style="width:245px;">Akorn Inc.</td>
			<td style="width:215px;">NDC 17478-215-05</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Goniovisc</td>
			<td style="width:245px;">BioVision Limited</td>
			<td style="width:215px;">NDC 17238-610-15</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Cyclopentolate Hydrochloride Ophthalmic Solution, USP&#160; 2%</td>
			<td style="width:245px;">Akorn Inc.</td>
			<td style="width:215px;">NDC 17478-097-10</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Gentamicin Sulfate Ointment USP, 0.1%</td>
			<td style="width:245px;">Perigo</td>
			<td style="width:215px;">NDC 45802-046-35</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Systane Ultra</td>
			<td style="width:245px;">Alcon Laboratories, Inc.</td>
			<td style="width:215px;">9006619-1013</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Tetracaine Hydrochloride</td>
			<td style="width:245px;">Bausch and Lomb</td>
			<td style="width:215px;">NDC 24208-920-64</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Ophthalmic Solution, USP 0.5%</td>
			<td style="width:245px;">Bausch and Lomb</td>
			<td style="width:215px;">NDC 24208-920-64</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">DPBS</td>
			<td style="width:245px;">Gibco Life Technologies</td>
			<td style="width:215px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Virus Preparations</td>
			<td style="width:245px;">ViGene /UNC</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Gold nanorods</td>
			<td style="width:245px;">NANOPARTz</td>
			<td style="width:215px;">D12M-850-1.75</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Fluorescein Sulfate AK-FLUOR 25%</td>
			<td style="width:245px;">Akorn Inc.</td>
			<td style="width:215px;">NDC 17478-250-20</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Coverslips</td>
			<td style="width:245px;">Fisher Scientific</td>
			<td style="width:215px;">12-548-A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Forceps</td>
			<td style="width:245px;">Milton</td>
			<td style="width:215px;">18-825</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Needles 30 guage</td>
			<td style="width:245px;">Beckton Dickenson&#160;&#160;</td>
			<td style="width:215px;">W11604</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Syringes</td>
			<td style="width:245px;">Beckton Dickenson&#160;&#160;</td>
			<td style="width:215px;">309659</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Bioptigen software Package</td>
			<td style="width:245px;">Bioptigen</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Proparacaine Hydrochloride Ophthalmic Solution, USP 0.5%</td>
			<td style="width:245px;">Akorn Inc.</td>
			<td style="width:215px;">NDC 17478-263-12</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Windows Excel</td>
			<td style="width:245px;">Microsoft</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Adobe Illustrator</td>
			<td style="width:245px;">Adobe&#160;</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Scale</td>
			<td style="width:245px;">Mettler</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Scissors</td>
			<td style="width:245px;">World Precision Instruments</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Ear punch</td>
			<td style="width:245px;">Nat&#8217;l band</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">CL 100 Light source</td>
			<td style="width:245px;">Welch Allyn</td>
			<td style="width:215px;">CL100</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Nitrogen Gas</td>
			<td style="width:245px;">Jackson Welding Supply</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Heated Water bath</td>
			<td style="width:245px;">Neslab</td>
			<td style="width:215px;">RTE-140</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Heating plate</td>
			<td style="width:245px;">In House</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Heating mat</td>
			<td style="width:245px;">Cincinnatti Sub Zero</td>
			<td style="width:215px;">273</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Clay mouse holder</td>
			<td style="width:245px;">Plast.i.clay American Art Clay Co.</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Betadine</td>
			<td style="width:245px;">MedLine&#160;</td>
			<td style="width:215px;">NDC53329-938-06</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Cotton Tip Applicators</td>
			<td style="width:245px;">American Health Service</td>
			<td style="width:215px;">Ctag</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">EtOH 70%</td>
			<td style="width:245px;">Fisher Scientific</td>
			<td style="width:215px;">BP2818-100</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Gloves Nitrile</td>
			<td style="width:245px;">VWR</td>
			<td style="width:215px;">89038-272</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Diagnosys ERG Color Dome instrument</td>
			<td style="width:245px;">Diagnosys Inc.</td>
			<td style="width:215px;">D125</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Contact lenses</td>
			<td style="width:245px;">In-house</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Diagnosys Software</td>
			<td style="width:245px;">Diagnosys Inc.</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Origin 6.1 software</td>
			<td style="width:245px;">OriginLab Corp.</td>
			<td style="width:215px;">N/A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:251px;">Reference electrodes</td>
			<td style="width:245px;">Ocuscience</td>
			<td style="width:215px;">F-Thread Electrode (DTL) 24&#8221;</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultrahigh resolution mouse optical coherence tomography to aid intraocular injection in retinal gene therapy research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Assessing the Presence, Rate and Uniformity of Model Outer Retinal Degeneration 
Measurements of the ONL were recorded from the OPL to the ELM, defining the limits of the ONL using the caliper tool provided in the instrument software. The goal was to map the progression of outer retinal degeneration in a partially humanized adRP mouse model. Comparable images from a control C57BL/6(J) mouse and an hC1/hC1//mWT/mWT mouse model, expressing two copies of the mutant huma...

Watch this Scientific Journal Video about Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research at JoVE.com

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

The overall goal of this subretinal injection procedure is to deliver a vector to the retina in a minimally invasive and reproducible manner that is observable in real-time. Hello, this method can be used to answer key questions in gene therapy research about vector delivery efficacy and therapeutic agent toxicity. Some advantages of this technique include minimal damage to the retina and other eye structures, precise injection site mapping, and rigorous quantitative in vivo measurements that allow solid statistical inference.For intraocular transgene injection, first place an anesthetized mouse onto the viewing stage of a retinal imaging system microscope and place the tips of a sterile blunt iris forceps at the seven and 10 o'clock positions of the eyelid while pushing the forceps open and down to gently induce proptosis. Use the forceps to coax the eyelids under the eye globe and direct the tip of the needle to about one to 1.5 millimeters below the edge of the corneal limbus. Next, apply a drop of sterile PBS solution to the eye and cover the eye with a sterile cover slip.Carefully drive the needle into the eye through the conjunctiva until a scleral depression is created. Use the micro manipulator to rotate the eye upward until the scleral depression is in focus, then drive the needle forward to create a sharp peak in the retina with the tip of the needle. Using the needle holder, rotate the needle just until the tip of the needle bores through the sclera and the RPE, allowing the flourescein in the tip of the needle to be visible under the retinal tissue in the immediate vicinity of the subretinal space and the retinal pigment epithelium cell monolayer.Drive the tip of the needle downward so it is tangential to the globe, then activate the injection pump with the foot pedal switch. After 0.5 to one microliters of transgene solution has been delivered to the subretinal space, withdraw the needle and confirm that the blood is stable and that fluid does not leak out of the injection tract site. To confirm the success of the injection, record a rectangular volume OCT and compare the images to images of the different kinds of potential injection outcomes.After confirmation of proper injection placement, use that rectangular OCT image to map the extent of the bleb onto the fundus image. To map the extent of the subretinal bleb, open a rectangular volume OCT image of the bleb such that a recognizable landmark within the eye is included in the image. Add as many calipers as necessary to identify the position of the left and right edges of the B scan, as well as the optic nerve head, ONH, if present, and the inflection point where the retina detaches from the RPE and choroid and then save the data.Then compile the saved files and plot the data, effectively creating a map of the injection bleb onto the en face OCT image. Overlay the plotted data onto the fundus image containing the injection site and save the merged image. Use this image to locate regions of interest during follow-up OCT imaging.To assess post injection retinal degeneration, first open a recorded high-resolution spectral domain OCT or HRSD OCT rectangular volume image and select a B scan to be measured. Magnify the selected image until it fills the screen and right click on the image. Click calipers to select the appropriate number of calipers, then use the configure caliper feature to assign the calipers as vertical in the angle block column and turn on the display caliper location to facilitate uniform caliper placement across the retina.Click apply and move each caliper to the appropriate location 0.1 to 0.2 millimeters apart across the B scan, taking care to place one caliper into the center of the optic nerve head when appropriate. When all of the calipers have been placed, click and drag each caliper to the length span of the region of interest, arbitrarily setting calipers not overlaying measurable regions of the B scan to maximum length. To measure the outer nuclear layer thickness, place the top of each caliper at the external limiting membrane and the bottom of each caliper at the bottom of the outer plexiform layer at 0.1 to 0.2 millimeter increments across the retina.After all of the calipers have been placed and adjusted to size, right click on the image and click save caliper data"then repeat the measurements as just demonstrated for every tenth B scan, keeping the calipers open and at the same location on the x-axis and adjust the lengths of the calipers as necessary, without moving them in the x-direction. When all of the scans have been measured, open the saved data files and click date modified"to arrange the files according to time saved. Open all of the files for each B scan measured and compile the raw data from each B scan into a single file from lowest to highest frame number and select the columns of data, including the caliper name, length, and center X columns.Ensure that the center X is the same for all of the B scans measured for each rectangular volume OCT image processed and delete any data from the calipers that were not used to record measurements. Set the caliper located at the center of the optic nerve head to zero and plot the data for X versus multiple Y data sets to get a 3D plot function for the thickness of the outer nuclear layer or any other measured retinal layer. Then compare the outer nuclear layer measurements between the control and experimental animals with corresponding ages to determine the rate and uniformity of any potential retinal degeneration.For 3D mapping of the retinal measurements, review the post-injection OCT images and note any identifiable landmarks, then position the animal to obtain a follow-up SDO CT image from the same region, taking care to include the same identifiable landmarks, and process the recorded images as just demonstrated. It is very important to obtain high-quality OCT images that include identifiable landmarks to facilitate reimaging of the previously injected retinal regions. In order to measure the changes in OS length, calipers were placed from the external limiting membrane to the Brooks membrane, allowing for reliable measurements since these layers are easily identified in OCT images.Differences in the measurements between these layers from different mouse lines were attributed to shorter outer segment lengths. If the subretinal injection is performed successfully, the opening of the subretinal space induces the production of a bleb that can be clearly visualized both in the en face view of the HRSD OCT imager and in the B scan images. Superimposing the border map of the injection site allows segmentation of the retinal tissue measurements and caliper position datasets to allow subsequent statistical testing of the effects of specific therapeutic agents on retinal degeneration.Once mastered, this technique can be completed in ten minutes if performed properly. While attempting this procedure it's important to remember to keep the surgical environment clean and sterile. After its development, this technique paved the way for researchers in the field of gene therapy to monitor the entire subretinal surgical injection process in real-time and created a way to immediately determine surgical success in vivo.Following this injection procedure, OCT and other methods like electro retinography can be performed to answer additional questions about transgene functional rescue and/or toxicity. After watching this video, you should have an understanding how to perform transscleral and transchoroidal subretinal injections, how to map the extent of the injection bleb onto the OCT from this image, and obtain rigorous measurements of the outer retinal layers. Don't forget that working with gene therapy vectors can be hazardous and that precautions, such as wearing the appropriate personal protective equipment should always be used while performing this procedure.

ultrahigh-resolution-mouse-optical-coherence-tomography-to-aid

Research

JoVE Journal

Medicine

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &amp;#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...